Search

This study aimed to improve the understanding of the nutrient modulation of Ostreopsis cf. ovata toxin content. During the 2018 natural bloom in the NW Mediterranean, the total toxin content (up to ca. 57.6 ± 7.0 pg toxin cell−1) varied markedly. The highest values often coincided with elevated O. cf. ovata cell abundance and with low inorganic nutrient concentrations. The first culture experiment with a strain isolated from that bloom showed that cell toxin content was higher in the stationary than in the exponential phase of the cultures; phosphate- and nitrate-deficient cells exhibited similar cell toxin variability patterns. The second experiment with different conditions of nitrogen concentration and source (nitrate, urea, ammonium, and fertilizer) presented the highest cellular toxin content in the high-nitrogen cultures; among these, urea induced a significantly lower cellular toxin content than the other nutrient sources. Under both high- and low-nitrogen concentrations, cell toxin content was also higher in the stationary than in the exponential phase. The toxin profile of the field and cultured cells included ovatoxin (OVTX) analogues -a to -g and isobaric PLTX (isoPLTX). OVTX-a and -b were dominant while OVTX-f, -g, and isoPLTX contributed less than 1-2%. Overall, the data suggest that although nutrients determine the intensity of the O. cf. ovata bloom, the relationship of major nutrient concentrations, sources and stoichiometry with cellular toxin production is not straightforward. [ABSTRACT FROM AUTHOR]

[Display omitted] • A LC-MS/MS method with microscale palytoxin oxidation was successfully validated. • High instrumental sensitivity and extract stability was achieved. • The quantitative method includes six palitoxin-like molecules in its scope. • In silico prediction provided solubility information about the main analytes. • Palytoxin ions were detected and confirmed in Haff disease-related samples. Haff disease typically develops after eating contaminated marine or freshwater species, especially fish. Despite still having an unknown etiology, recent reports have suggested its possible correlation with palytoxins. Therefore, the present work aimed to optimize and perform a validation of a sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of palytoxin and some of its analogs, with the main purpose of investigating their presence in marine and freshwater food samples associated with Haff disease in Brazil. The method optimization was performed using a central composite rotatable design and fish samples fortified with the palytoxin standard. Then, the optimized method was validated for different food matrices, including freshwater and marine fish, mollusks, and crustaceans. The sample preparation involved a solid–liquid extraction using methanol and water, solid-phase extraction using Strata-X cartridges, and on-column palytoxin oxidation. The detection of the main oxidized fragments (amino and amide aldehydes) was achieved by LC-MS/MS with electrospray ionization in positive mode, using a C18 column, as well as acetonitrile and water as mobile phases, both acidified with 0.1 % of formic acid. After optimization and validation, the etiological investigation involved the analysis of 16 Brazilian Haff disease-related food samples (in natura and leftover meals) from 2022. The method was demonstrated to be appropriate for quantitative analysis of freshwater and marine species. So far, it has proven to be one of the most sensitive methods related to palytoxin detection (LOD 10 μg/kg), being able to work in a range that includes the provisional ingestion limit (30 μg/kg). Regarding the Haff disease-related samples analysis, there is a strong indication of palytoxin contamination since the amino aldehyde (common fragment for all palytoxins) was detected in 15 of the 16 samples. Selected results were confirmed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). [ABSTRACT FROM AUTHOR]

Blooms of the benthic toxic dinoflagellate genus Ostreopsis have been recorded more frequently during the last two decades, particularly in warm temperate areas such as the Mediterranean Sea. The proliferation of Ostreopsis species may cause deleterious effects on ecosystems and can impact human health through skin contact or aerosol inhalation. In the eastern Atlantic Ocean, the toxic O. cf. ovata has not yet been reported to the north of Portugal, and the only species present further north was O. cf. siamensis, for which the toxic risk is considered low. During summer blooms of unidentified Ostreopsis species on the French Basque coast (Atlantic) in 2020 and 2021, people suffered from irritations and respiratory disorders, and the number of analyzed cases reached 674 in 2021. In order to investigate the causes, sampling was carried out during summer 2021 to (i) taxonomically identify Ostreopsis species present using a molecular approach, (ii) isolate strains from the bloom and culture them, and (iii) characterize the presence of known toxins which may be involved. For the first time, this study reports the presence of both O. cf. siamensis and O. cf. ovata, for which the French Basque coast is a new upper distribution limit. Furthermore, the presence of ovatoxins a, b, c, and d in the environmental sample and in a cultivated strain in culture confirmed the toxic nature of the bloom and allowed identifying O. cf. ovata as the producer. The present data identify a new health risk in the area and highlight the extended distribution of some harmful dinoflagellates, presumably in relation to climate change. [ABSTRACT FROM AUTHOR]

Recurrent blooms of the toxic dinoflagellate Ostreopsis cf. ovata are frequently reported in the Northwestern Mediterranean Sea. The impact of these proliferations on other microalgal species inhabiting the same habitats is of interest from an ecological prospective. In vitro experiments were carried out to investigate the influence of O. cf. ovata on the growth of the co-occurring benthic diatoms Licmophora paradoxa, Navicula arenaria and the benthic dinoflagellates Prorocentrum lima and Coolia monotis. Overall, O. cf. ovata exhibited weak allelopathic effects towards these microalgal species, with a reduction in the cell abundance for L. paradoxa and P. lima only. Interestingly, dead cells of L. paradoxa and N. arenaria were observed embedded in the thick mucus surrounding O. cf. ovata cells, suggesting that the mucous layer could act as a toxic phycosphere, especially for non-motile cells. All competitors were further exposed for 24 h to ovatoxins, the major toxins produced by O. cf. ovata, and the maximum quantum yield efficiency of L. paradoxa, N. arenaria and P. lima was affected at a minimum concentration of 10 µg mL−1. We then hypothesized that the diffusion of solubilized ovatoxins in the culture medium affects only moderately the competitors' growth, whereas their accumulation in the mucus would yield deleterious effects. More precisely, the competitors' sensitivity to ovatoxins was enhanced in their stationary phase of growth and resulted from a rapid inhibition of an uncharacterized photosynthetic step downstream photosystem II. Altogether, these results emphasize the predominant role of the O. cf. ovata's mucus in driving ecological interactions and suggest that it can affect the growth of several benthic microalgae by accumulating the potent ovatoxins. [ABSTRACT FROM AUTHOR]

This study aimed to improve the understanding of the nutrient modulation of Ostreopsis cf. ovata toxin content. During the 2018 natural bloom in the NW Mediterranean, the total toxin content (up to ca. 57.6 ± 7.0 pg toxin cell−1) varied markedly. The highest values often coincided with elevated O. cf. ovata cell abundance and with low inorganic nutrient concentrations. The first culture experiment with a strain isolated from that bloom showed that cell toxin content was higher in the stationary than in the exponential phase of the cultures; phosphate- and nitrate-deficient cells exhibited similar cell toxin variability patterns. The second experiment with different conditions of nitrogen concentration and source (nitrate, urea, ammonium, and fertilizer) presented the highest cellular toxin content in the high-nitrogen cultures; among these, urea induced a significantly lower cellular toxin content than the other nutrient sources. Under both high- and low-nitrogen concentrations, cell toxin content was also higher in the stationary than in the exponential phase. The toxin profile of the field and cultured cells included ovatoxin (OVTX) analogues -a to -g and isobaric PLTX (isoPLTX). OVTX-a and -b were dominant while OVTX-f, -g, and isoPLTX contributed less than 1-2%. Overall, the data suggest that although nutrients determine the intensity of the O. cf. ovata bloom, the relationship of major nutrient concentrations, sources and stoichiometry with cellular toxin production is not straightforward.

Blooms of the dinoflagellate Ostreopsis cf. ovata are regularly associated with human intoxications that are attributed to ovatoxins (OVTXs), the main toxic compounds produced by this organism and close analogs to palytoxin (PlTX). Unlike for PlTX, information on OVTXs'toxicity are scarce due to the absence of commercial standards. Extracts from two cultures of Mediterranean strains of O. cf. ovata (MCCV54 and MCCV55), two fractions containing or not OVTXs (prepared from the MCCV54 extract) and OVTX-a and -d (isolated from the MCCV55 extract) were generated. These chemical samples and PlTX were tested on a panel of cell types from several organs and tissues (skin, intestine, lung, liver and nervous system). The MCCV55 extract, containing a 2-fold higher amount of OVTXs than MCCV54 extract, was shown to be more cytotoxic on all the cell lines and more prone to increase interleukin-8 (IL-8) release in keratinocytes. The fraction containing OVTXs was also cytotoxic on the cell lines tested but induced IL-8 release only in liver cells. Unexpectedly, the cell lines tested showed the same sensitivity to the fraction that does not contain OVTXs. With this fraction, a pro-inflammatory effect was shown both in lung and liver cells. The level of cytotoxicity was similar for OVTX-a and –d, except on intestinal and skin cells where a weak difference of toxicity was observed. Among the 3 toxins, only PlTX induced a pro-inflammatory effect mostly on keratinocytes. These results suggest that the ubiquitous Na+/K+ ATPase target of PlTX is likely shared with OVTX-a and -d, although the differences in pro-inflammatory effect must be explained by other mechanisms. [Display omitted] • Extracts of Ostreopsis cf. ovata were cytotoxic to a broad range of cell lines. • The fraction not containing ovatoxins induced IL-8 release on liver and lung cells. • PlTX was more cytotoxic than OVTX-a and –d on cells from intestine and the nervous system. • A similar IC 50 was observed on lung cells for the three toxins. • OVTX-a and –d did not induce IL-8 release on intestinal, lung and skin cells. [ABSTRACT FROM AUTHOR]

Blooms of the benthic toxic dinoflagellate genus Ostreopsis have been recorded more frequently during the last two decades, particularly in warm temperate areas such as the Mediterranean Sea. The proliferation of Ostreopsis species may cause deleterious effects on ecosystems and can impact human health through skin contact or aerosol inhalation. In the eastern Atlantic Ocean, the toxic O. cf. ovata has not yet been reported to the north of Portugal, and the only species present further north was O. cf. siamensis, for which the toxic risk is considered low. During summer blooms of unidentified Ostreopsis species on the French Basque coast (Atlantic) in 2020 and 2021, people suffered from irritations and respiratory disorders, and the number of analyzed cases reached 674 in 2021. In order to investigate the causes, sampling was carried out during summer 2021 to (i) taxonomically identify Ostreopsis species present using a molecular approach, (ii) isolate strains from the bloom and culture them, and (iii) characterize the presence of known toxins which may be involved. For the first time, this study reports the presence of both O. cf. siamensis and O. cf. ovata, for which the French Basque coast is a new upper distribution limit. Furthermore, the presence of ovatoxins a, b, c, and d in the environmental sample and in a cultivated strain in culture confirmed the toxic nature of the bloom and allowed identifying O. cf. ovata as the producer. The present data identify a new health risk in the area and highlight the extended distribution of some harmful dinoflagellates, presumably in relation to climate change.

The genus Ostreopsis includes some species that produce high biomass blooms and/or synthesize toxic compounds that can be transferred through the marine food webs or aerosolized causing ecological, human health and socio-economic impacts. Ostreopsis species are increasing their biogeographic distribution from tropical to more temperate waters and causing recurrent blooms in certain coastal areas, thus constituting an emerging concern worldwide. The proliferation capacity of Ostreopsis is due to a complex and poorly understood combination of multiple factors, and may be a paradigm of chemical ecology reviewed here. A first section summarizes the basic knowledge on the different Ostreopsis species, the toxins they produce and the described foodborne and airborne effects of Ostreopsis toxins on humans. Secondly, direct and indirect interactions between Ostreopsis species and their environment are reviewed. Mucopolysaccharide substances produced by the cells to attach to different substrates appear to be a key element on the chemical ecology and requires further study. However, this research is challenged by technical limitations to conduct ecologically realistic and harmonized studies where organisms can be in direct contact with Ostreopsis cells, their mucus and/or the released extracellular toxic compounds. Understanding the transfer mechanisms of these substances within the food web, potentially affecting humans is critical and requires further study with new analytical tools. Still, the progress in knowledge achieved in the last years, combined with experimental and field studies using cutting edge methods will facilitate to address the open questions on the chemical ecology of Ostreopsis and understand its bloom dynamics now, and under future climate and anthropogenic change scenarios.

Recurrent blooms of Ostreopsis cf. ovata have been reported in Brazil and the Mediterranean Sea with associated ecological, and in the latter case, health impacts. Molecular data based on the D1−D3 and D8−D10 regions of the LSU rDNA and ITS loci, and the morphology of O. cf. ovata isolates and field populations from locations along the Brazilian tropical and subtropical coastal regions and three oceanic islands are presented. Additional ITS sequences from three single cells from the tropical coast are provided. Toxin profiles and quantities of PLTX and their analogues; OVTXs; contained in cells from two clonal cultures and two field blooms from Rio de Janeiro were investigated. Morphology was examined using both light and epifluorescence microscopy. Morphometric analysis of different strains and field populations from diverse locations were compared. Molecular analysis showed that six of the seven sequences grouped at the large “Atlantic/Mediterranean/Pacific” sub-clade, while one sequence branched in a sister clade with sequences from Madeira Island and Greece. The toxin profile of strains and bloom field samples from Rio de Janeiro were dominated by OVTX-a and -b, with total cell quotas (31.3 and 39.3 pg cell−1) in the range of that previously reported for strains of O. cf. ovata.

The toxin profile and hemolytic activity of a strain of Ostreopsis cf. ovata (UFBA013) isolated from Todos os Santos Bay (northeastern Brazil) were evaluated under different levels of N and P. Phylogenetic analyses based on ITS rDNA region (ITS1-5.8S-ITS2) placed UFBA013 within the Atlantic/Mediterranean/Pacific clade of O . cf. ovata . Growth experiments were conducted in f/2 medium modified by adding N and P (P: 0–36 μM; N: 0–882 μM). The growth kinetics was adequately described by logistic equations. The best growth (highest G m ) was recorded under levels of N/P = 0/18, 129/5 and 441/36, while one of the lowest G m was obtained under P-depletion. The maximum and specific maximum growth rates (as v m; cells mL −1 d −1 and μ m ; d −1 ) were achieved with N limitation (N/P = 441/36) and P-limitation/depletion (753/5.3 and 441/0) and are the highest values reported in the literature, most similar to isolates from Pacific and Mediterranean areas. The control experiment (N/P = 441/18) also yielded similar values to those from some Mediterranean isolates, but higher than formerly reported for Brazilian isolates. In all conditions assayed, no palytoxin (PLTX) was detected. The ovatoxins (OVTXs) a, b, c, d and e did not show significant differences in cell quota between exponential and stationary phases. A significant relationship was detected between OVTXs concentration and hemolytic activity. [ABSTRACT FROM AUTHOR]

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg−1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg−1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell−1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]3+. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg−1 for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg−1 in mussel, cockle, and ensis, and at 60 µg PlTX kg−1 in oyster.

Intense blooms of the toxic dinoflagellate Ostreopsis have been a recurrent phenomenon along several Mediterranean coasts. Blooms have been associated with noxious effects on human health and mortality of marine organisms, due to the production of palytoxin-like compounds. We analyzed the toxin concentrations throughout an O. cf. ovata bloom to highlight their relationships with environmental parameters in the Conero Riviera, northern Adriatic Sea. High temperature and balanced nutrient conditions were the optimal environmental conditions to start and sustain blooms as well as to maximize toxin production. Ostreopsis showed a gradual decrease of toxin content throughout the bloom ascribed to the occurring of the same non-optimal conditions that led to the bloom decline. Moreover, our results suggest that toxin fraction released during bloom could be higher than that released in batch culture. Results from this study pointed out that the first bloom phase is potentially the most dangerous to human health. [ABSTRACT FROM AUTHOR]

The genus Ostreopsis includes several toxic species that can develop blooms in benthic ecosystems, with potential harmful consequences for human health and marine invertebrates. Despite of this, little is known about the allelopathic interactions between these organisms and other co-occurring microalgae that exploit similar spatial and nutrient resources in benthic ecosystems. The aim of this study was to follow these interactions in cultures of two Ostreopsis ribotypes with different toxin profiles ( O . cf. ovata contained ovatoxins-a, b, c and e, while only ovatoxin-d was found in O . sp. “ Lanzarote-type”), mixed with species of three benthic dinoflagellate genera ( Coolia , Prorocentrum and Gambierdiscus ), isolated from the same area (North East Atlantic, Canary Islands). In a first experiment, the potential allelopathic effects on growth rates were followed, in mixed cultures of Coolia monotis (a non toxic species) exposed to the clarified medium and to cells of O . sp. “ Lanzarote-type” and O . cf. ovata . Growth delayed in C. monotis was observed specially in clarified medium, while the O. sp. “ Lanzarote-type” strain attained much lower densities in mixed cultures. In a second experiment, we examined the potential effects of clarified media from O. sp. “ Lanzarote-type” and O . cf. ovata on the adherence capacity in two toxic species ( Prorocentrum hoffmannianum and Gambierdiscus excentricus ). Contrasting effects were found: a significant increase of adherence capacity in P. hoffmannianum vs attachment decline in G. excentricus , that experienced also severe deleterious effects (cell lysis). Our results suggest the existence of weak to moderate allelopathic interactions between the studied organisms, although the outcome is dependent on the species involved. [ABSTRACT FROM AUTHOR]

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3′, 7″, 6c, 7s, 5‴, 2′′′′, is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1′ and 3′ makes them approximately hexagonal, (ii) the 1′ plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6″. The round thecal pores are bigger than the other two Mediterranean species ( O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus ( DV: 60.07 ± 5.63 μm, AP: 25.66 ± 2.97 μm, W: 39.81 ± 5.05 μm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX-a and structural isomers OVTX-d and -e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin-like compounds (ovatoxin-i, ovatoxin-j1, ovatoxin-j2, and ovatoxin-k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28-0.94 pg · cell−1. On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C-31.5°C), with peaks in June and August. [ABSTRACT FROM AUTHOR]

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata are usually associated with shallow and calm coastal waters, characterized by low nutrient concentrations. The algal cells typically cover the benthic substrates, such as the macroalgal and invertebrate communities and rocks, forming a mucilaginous film. Data reported on O . cf. ovata toxin production observed under both field and culture conditions show high variability in terms of toxic profile and cellular content; little is known about the environmental and physiological aspects which regulate the toxin dynamics. In this study, O . cf. ovata physiology was investigated using batch cultures supplied with nutrient concentrations similar to those found in the Adriatic Sea during the recurrent blooms and the observed cellular dynamics were compared with those found in a culture grown under optimal conditions, used as a reference. Data on the cellular C, N and P content during the growth highlighted a possible important role of the cellular nutritional status in regulating the toxin production that resulted to be promoted under specific intervals of the C:N and C:P ratios. The variable toxicity found for O . cf. ovata in various geographic areas could be related to the different in situ prevalent environmental conditions (e.g., nutrient concentrations) which affect the cellular elemental composition and carbon allocation. The obtained results strongly suggest that in the environment toxin production is steadily sustained by a low and constant nutrient supply, able to maintain appropriate cellular C:N (>12) or C:P (>170) ratios for a long period. These results explain to some extent the variability in toxicity and growth dynamics observed in blooms occurring in the different coastal areas. [ABSTRACT FROM AUTHOR]

Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island were analyzed through an integrated approach based on molecular, chemical, and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j, ovatoxin-j, and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in an additional CHO moiety and an unsaturation in the region C49-C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j and OVTX-k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and OVTX-j). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg cell, definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island poses a relatively low risk to humans. [ABSTRACT FROM AUTHOR]

Ostreopsis cf. ovata is a benthic marine dinoflagellate, which blooms in summer along the coasts of the Mediterranean Sea, including many Italian regions since early 1990s. In tropical areas O. cf. ovata produces mainly palytoxins whereas in the Mediterranean basin it produces ovatoxins, and only trace amounts of a putative palytoxin. The toxicological profile of ovatoxins is still unknown. This paper describes the update of the Italian guidelines for the management of O. cf. ovata blooms related to bathing and recreational activities. The rationale for the guidelines starts from the current scientific knowledge on chemical, toxicological, and epidemiological aspects and on its diffusion, which are briefly summarized in the first part of the paper. On this basis a risk-based approach is developed, with the purpose to protect people from oral, dermal and inhalation exposure (the ones that so far caused major health problems), taking into account also the weather and sea conditions, which are crucial for the onset of blooms. Accordingly, the guidelines provide information on what indicators are needed to set up environmental monitoring activities and actions to take during diverse phases. They also outline a multi-step health surveillance system, and a system of communication to the public. Finally the still important scientific gaps and research needs are highlighted. [ABSTRACT FROM AUTHOR]

Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14-1000 ng mL in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC-HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 μg kg in crude mussel extracts and 15 μg kg after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this. [ABSTRACT FROM AUTHOR]

Human intoxications in the Mediterranean Sea have been linked to blooms of the dinoflagellate Ostreopsis cf. ovata, producer of palytoxin (PlTX)-like toxins called ovatoxins (OVTXs). Exposure routes include only inhalation and contact, although PlTX-poisoning by seafood has been described in tropical regions. To address the impact of OVTXs on the intestinal barrier, dinoflagellate extracts, purified OVTX-a and -d and PlTX were tested on differentiated Caco-2 cells. Viability, inflammatory response and barrier integrity were recorded after 24 h treatment. OVTX-a and -d were not cytotoxic up to 20 ng/mL but increased IL-8 release, although to a lesser extent compared to PlTX. While PlTX and OVTX-a (at 0.5 and 5 ng/mL respectively) affected intestinal barrier integrity, OVTX-d up to 5 ng/mL did not. Overall, OVTX-d was shown to be less toxic than OVTX-a and PlTX. Therefore, oral exposure to OVTX-a and -d could provoked lower acute toxicity than PlTX., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Abstract: Since 1998, blooms of the epi-benthic dinoflagellate, Ostreopsis cf. ovata Fukuyo have been reported in the Arraial do Cabo area, Rio de Janeiro, Brazil. This dinoflagellate can produce one of the most lethal marine toxins, palytoxin (PLTX) and its analogues. In the study reported herein, the growth rate, cellular dimensions and toxin profile of two O. cf. ovata strains isolated from Armação dos Búzios, Rio de Janeiro were investigated. Molecular analysis (5.8S rDNA gene and ITS regions) of one strain was also performed. Respectively, strains LCA-E7 and LCA-B7 showed growth rates of 0.15 and 0.10divday−1, and cell dimensions of the strains and field population were similar to those found for O. ovata from the Mediterranean Sea. Ribosomal DNA genetic sequences obtained from the nuclear region (ITSs and 5.8S) of strain LCA-E7 confirmed that this is O. cf. ovata and grouped this isolate in a robust clade with isolates from Brazil and the Mediterranean. The production of putative (p)PLTX and ovatoxin (OVTX) was assessed using ouabain inhibited hemolytic assays. Extracts of the two strains caused hemolysis and was positively related to O. cf. ovata cell numbers. Analysis by liquid chromatography tandem mass spectrometry identified OVTX-a, -b, -c, -d and -e. Estimated concentrations varied between 20 and 171pg[OVTX-a]cell−1, 23 and 205pg[OVTX-b]cell−1, 3 and 37pg[OVTX-c]cell−1 and 3 and 80pg[OVTX-d+e]cell−1. OVTX-a and -b were the major components of the toxin profile produced by strains LCA-E7 and LCA-B7 and represented between 45 and 95% of the total OVTX+pPLTX content of these cells. Concentrations of OVTXs produced by the Brazilian strains were higher than those found in an O. cf. ovata Adriatic isolate, although in the same magnitude. [Copyright &y& Elsevier]

Abstract: In the Mediterranean Sea, blooms of Ostreopsis cf. ovata and Ostreopsis siamensis have become increasingly frequent in the last decade and O. cf. ovata was found to produce palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, -d and -e), a class of highly potent toxins. The environmental conditions seem to play a key role in influencing the abundance of Ostreopsis spp. High cell densities are generally recorded in concomitance with relatively high temperature and salinity and low hydrodynamics conditions. In this study the effects of temperature and salinity on the growth and toxicity of an Adriatic O. cf. ovata isolate were investigated. The highest growth rates of the Adriatic strain were recorded for cultures grown at 20 °C and at salinity values of 36 and 40, in accordance with natural bloom surveys. Toxicity was affected by growth conditions, with the highest toxin content on a per cell basis being measured at 25 °C and salinity 32. However, the highest total toxin content on a per litre basis was recorded at 20 °C and salinity 36, since under such conditions the growth yield was the highest. O. cf. ovata had lethal effects on Artemia nauplii and juvenile sea basses, and produced haemolysis of sheep erythrocytes. A comparison between haemolysis neutralization assay and HR LC-MS results showed a good correlation between haemolytic effect and total toxin content measured through HR LC-MS. Considering the increasing need for rapid and sensitive methods to detect palytoxin in natural samples, the haemolytic assay appears a useful method for preliminary quantification of the whole of palytoxin-like compounds in algal extracts. [Copyright &y& Elsevier]

Abstract: Since its isolation dated back to as far as 1971, palytoxin has all along drawn scientists’ attention from across the world because of its high toxicity and fascinating chemical architecture. Commitment of the international scientific community to the study of this extremely potent non-proteic toxin has led to discover quite a number of palytoxin analogues. Once confined only to tropical and subtropical areas, palytoxins have recently spread also to more temperate regions, such as the Mediterranean Sea where they have caused severe human intoxications. Studies on the Mediterranean toxic outbreaks brought to light the existence of further palytoxin-like compounds, ovatoxins, never reported elsewhere in the world. [Copyright &y& Elsevier]

Abstract: The state of the art of LC-MS of palytoxin and its analogues is reported in the present review. MS data for palytoxin, 42-hydroxy-palytoxin, ostreocin-D, mascarenotoxins, and ovatoxins, obtained using different ionization techniques, namely fast-atom bombardment (FAB), matrix assisted laser desorption ionization (MALDI), and electrospray ionization (ESI), are summarized together with the LC-MS methods used for their detection. Application of the developed LC-MS methods to both plankton and seafood analysis is also reported, paying attention to the extraction procedures used and to limits of detection (LOD) and quantitation (LOQ) achieved. In a research setting, LC-MS has shown a good potential in determination of palytoxin and its analogues from various sources, but, in a regulatory setting, routine LC-MS analysis of palytoxins is still at a preliminary stage. The LOQ currently achieved in seafood analysis appears insufficient to detect palytoxins in shellfish extract at levels close to the tolerance limit for palytoxins (30 μg/kg) proposed by the European Food Safety Authority (). In addition, lacking of certified reference standard of palytoxins as well as of validation studies for the proposed LC-MS methods represent important issues that should be faced for future perspectives of LC-MS technique. [Copyright &y& Elsevier]

In the dinophyte genus Ostreopsis, seven out of 11 described species are known to produce various toxic compounds that were characterized in the palytoxins family. Species in the genus shared identical thecal plate patterns but differed in size, shape, and thecal plate ornamentation. Two species, O. cf. ovata and O. siamensis, have been reported from the Western Pacific, but information on toxin production is scarce. Here, we established nine strains of Ostreopsis from six localities in the South China Sea (SCS), covering the Gulf of Thailand, northern SCS (Hainan Island, Beibu Bay), and southern SCS (Peninsular Malaysia). Their morphology was examined by light and electron microscopy and the molecular phylogeny was inferred based on the LSU rDNA (D1-D3) and ITS rDNA sequences using maximum likelihood and Bayesian inference. Both O. cf. ovata and O. siamensis, albeit morphologically closely related, can be distinguished by a feature of the thecal pores with pronounced ridges in the latter. Molecular data further supported their species identity. Toxin production in the strains was examined by LC-MS/MS. O. cf. ovata strain T5PRBost02 was observed to produce Ovatoxin-k and Ovatoxin-j2 only; while Ostreocin-B and Ostreocin-D was produced by O. siamensis strain T10PRBost04. This is the first report confirming the production of palytoxins analogs in Ostreopsis species from the region., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ostreopsis cf. ovata is a benthic and ovatoxin-producing dinoflagellate proliferating yearly along the Mediterranean coasts where blooms have been related to human illness and unusual mortality of marine organisms. The spreading of O. cf. ovata in this temperate area has been linked to global changes and its consequences such as the increase of temperature or light intensities. In the present study, an experimental design using batch cultures of pre-acclimated cells of a strain of O. cf. ovata isolated from Villefranche-sur-Mer (NW Mediterranean Sea, France), was implemented to investigate the combined effect of temperature (23, 27 and 30 °C) and light intensity (200, 400 and 600 µmol m -2 s -1 ) on the growth, metabolome and OVTX content. Both light intensity and temperature affected the growth as significantly higher growth rates were obtained under 400 and 600 µmol m -2 s -1 while the maximum values were obtained at 27 °C (0.48 d -1 ). Metabolomic analyses highlighted a clear effect only for temperature that may correspond to two different strategies of acclimation to suboptimal temperatures. Significant features (such as carotenoid and lipids) modified by the temperature and/or light conditions were annotated. Only temperature induced a significant change of OVTX content with higher values measured at the lowest temperature of 23 °C (29 - 36 pg cell -1 ). In a context of global changes, these results obtained after acclimation suggest that the increase of temperature might favor the proliferation of less toxic cells. However, in the light of the intraspecific variability of O. cf. ovata, further studies will be necessary to test this hypothesis. This study also highlighted the lack of knowledge about the metabolome composition of such non-model organisms that impairs data interpretation. There is a need to study more deeply the metabolome of toxic dinoflagellates to better understand how they can acclimate to a changing environment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Over the last fifteen years, blooms of the genus Ostreopsis have been reported more frequently and at higher abundances in the Mediterranean area. Ostreopsis cf. ovata is known to produce ovatoxins (OVTXs), structural analogues of palytoxin, which is one of the most potent non-polymeric toxins. However, the production of OVTXs is poorly characterized in situ. The present study focuses on toxin content and profile according to the bloom phase during summer 2017 in Villefranche-sur-Mer, France (NW Mediterranean Sea), depth (from 0.5 to 5 m) and three different macroalgal substrates of this epiphytic dinoflagellate (Padina pavonica, Dictyota spp. and Halopteris scoparia). Ovatoxin quantification of all samples was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The bloom started at the end of June and declined in mid-July, showing the typical seasonal pattern of the NW Mediterranean Sea area. The peak was observed on the 10 July with 1.8 × 10 6 cells/g FW and 1.7 × 10 4 cells/L for benthic and planktonic cells, respectively. Total toxin content of cells, collected using artificial substrates, increased during the exponential and stationary growth phases. After reaching a maximum concentration of 9.2 pg/cell on 18 July, toxin concentration decreased and remained stable from 25 July until the end of monitoring. A decreasing trend of the abundance and of the associated total toxin content was noted with depth. Finally, the decreasing order of maximal epiphytic concentration of O. cf. ovata was: Dictyota spp. (8.3 × 10 5 cells/g FW), H. scoparia (3.1 × 10 5 cells/g FW) and P. pavonica (1.6 × 10 5 cells/g FW). Interestingly, the highest OVTX quota was obtained in cells present on Halopteris scoparia, then on Dictyota spp. and Padina pavonica. This suggests that the nature of the macroalgal substrate influences both growth and toxin production of O. cf. ovata and further work will be required to understand the underlying mechanisms (e.g., competition for nutrition, pH or allelopathic interaction). However, the toxin profiles (i.e., the proportion of each ovatoxin analogue) were not affected by any of the studied parameters (bloom phase, depth, macroalgae or artificial substrates)., (Copyright © 2019 Elsevier B.V. All rights reserved.)